Widespread trypanosome infections in a population of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in Virginia, USA

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1 Widespread trypanosome infections in a population of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in Virginia, USA Andrew K. Davis & William A. Hopkins Parasitology Research Founded as Zeitschrift für Parasitenkunde ISSN Volume 112 Number 1 Parasitol Res (2013) 112: DOI /s

2 Parasitol Res (2013) 112: DOI /s SHORT COMMUNICATION Widespread trypanosome infections in a population of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in Virginia, USA Andrew K. Davis & William A. Hopkins Received: 22 March 2012 /Accepted: 6 August 2012 /Published online: 25 August 2012 # Springer-Verlag 2012 Abstract Eastern hellbender salamanders (Cryptobranchus alleganiensis alleganiensis) are declining in North America and because of this the health status of individuals in several populations is closely monitored by researchers. During a health survey of hellbenders from a stream in Smyth County, VA, USA, we examined Giemsa-stained blood smears of 71 animals captured during 2011 for the presence of blood parasites. We discovered an unknown species of trypanosome that was apparently widespread within this population; 40 of the 71 individuals (56.3 %) were infected. There are seven known trypanosome species of caudate amphibians; based on microscopic examination, the parasite we observed appeared most similar to Trypanosoma cryptobranchi, which was reported in this species only once before, 76 years ago, from a single animal apparently captured in Iowa. Given that some trypanosomes can adversely affect the health of their hosts, we recommend further monitoring be done in this and other hellbender populations to determine the geographic extent of the parasite and its effects on its increasingly rare host. Introduction In the Eastern United States, populations of hellbenders (Cryptobranchus alleganiensis), a large aquatic salamander A. K. Davis (*) Odum School of Ecology, University of Georgia, Athens, GA 30602, USA akdavis@uga.edu W. A. Hopkins Department of Fish and Wildlife Conservation, Virginia Tech, Blacksburg, VA 24061, USA that lives in rocky streams, are declining (Wheeler et al. 2003; Foster et al. 2009; Burgmeier et al. 2011a). This is true for the eastern form (Cryptobranchus alleganiensis alleganiensis) which has a range encompassing much of the Eastern United States and the Ozark hellbender (Cryptobranchus alleganiensis bishopi), which is restricted to a narrow range in Missouri and Arkansas. In fact, the Ozark hellbender was recently classified as an endangered species by the US Fish and Wildlife Service (Federal Register 2011). Because of these population declines, numerous investigations have evaluated the health status of individuals from populations of both subspecies. These include examination of blood cell counts and morphology, hematology and serum chemistry, heavy metal loads, sperm quality, stress hormone levels, immunological responses, disease testing, and blood parasite prevalence (Jerrett and Mays 1973; Solis et al. 2007; Huang et al. 2010; Burgmeier et al. 2011b; Hopkins and DuRant 2011). With regard to blood parasites, in most cases, few have been reported. One notable exception is a recent report of unidentified blood parasites from Ozark hellbenders (Huang et al. 2010), which appeared to be trypanosomes (J. Briggler, personal communication), although they were not examined in detail in that study. Recently, we examined blood films from a population of eastern hellbenders from Virginia and discovered infections of trypanosomes; we describe these parasites in this paper. There are seven species of trypanosomes that have been reported in caudate amphibians (Woo and Bogart 1986). Only one has been reported in hellbenders; in a very early account, blood from two eastern hellbenders was examined and one individual was infected with a trypanosome species (Roudabush and Coatney 1937). Roudabush and Coatney provided basic morphological measurements as well as line drawings and named the parasite Trypanosoma cryptobranchi. Later, Lehmann (1959) examined the same blood film

3 454 Parasitol Res (2013) 112: and provided further details about the morphology of T. cryptobranchi and how it is distinguished from other trypanosomes in salamanders. A key characteristic is the appearance and position of the kinetoplast, which varies in shape and placement among species of salamanders, as it does across other vertebrate taxa (Clark and Wallace 1960). In T. cryptobranchi, it is rod-shaped or elliptical and located near the posterior end (15 % of the body length from it). Another key characteristic is the centrally located nucleus and the lack of endosome (Lehmann 1959). In the two other trypanosome species that have rod-shaped kinetoplasts (Trypanosoma granulosae and Trypanosoma ambystomae), the nucleus is not centrally located in the body, and in T. granulosae, the endosome is consistently visible. Furthermore, Roudabush and Coatney (1937) stated that the kinetoplast in T. cryptobranchi is typically perpendicular to the long axis of the parasite body. location. The animals were all captured by hand (i.e., snorkeling) from under the rocks and processed in the field, which included basic morphometrics, determining sex (based on the presence of the swollen cloaca of males), implanting a pit tag for later identification, as well as taking a blood sample. For this, blood was drawn from the caudal artery with a 26-gauge needle, and a drop was smeared on a clean microscope slide for blood parasite screening. Slides were air-dried and returned to the lab. All field procedures were approved by the Virginia Tech Animal Care and Use Committee (VT IACUC # FIW). In the lab, all slides were stained with a buffered Wright Giemsa stain (Camco Quik Stain II) and examined with a light microscope, at 400, by a single observer (AKD). For each slide, 50 random fields of view were examined for the presence of blood parasites. If no parasites were found, the animal was assumed to be uninfected. Material and methods Hellbenders were captured between June and August 2011 from a stream in Smyth County, VA, USA as part of an ongoing effort to monitor the health of hellbenders in this Results and discussion We examined blood films from a total of 71 hellbenders. We were able to confirm the sex of 63 individuals: 31 were females and 32 were males. We noted trypanosome Fig. 1 Photomicrographs of trypanosomes (presumed to be T. cryptobranchi) observed in eastern hellbenders from Virginia (a e) as well as line drawings from the original description of T. cryptobranchi (f) from Roudabush and Coatney (1937). Small arrows point to the kinetoplast, which is rod-shaped or elliptical in T. cryptobranchi. Also note the centrally located nucleus in all cases. The free flagellum was never seen, and an undulating membrane was rarely seen in the Virginia specimens (thick arrow in B)

4 Parasitol Res (2013) 112: infections in 40 hellbenders (56.3 %) overall. Seventeen females (54.8 %) were infected while 19 males (59.4 %) were infected with trypanosomes. At the magnification used ( 400), the average number of trypanosome parasites observed in all infected animals was 2.0 (3.1 SD) in 50 fields of view (range01 14). Parasitemia level was not statistically different between males and females (two-sampled t test, df034, t0 1.56, p00.126). Based on our observations and measurements, the parasite appeared most similar (with some exceptions, indicated below) to the description of T. cryptobranchi given by Roudabush and Coatney (1937). It was elongate, often with a corkscrew-like appearance, was bluish-purple under Giemsa staining, had a rod-shaped, pink-staining kinetoplast located near the posterior end, and a centrally located nucleus (Fig. 1a e). In most cases, the long axis of the nucleus was oriented parallel to the longitudinal axis of the trypanosome body, whereas the kinetoplast was perpendicular to the long axis of the parasite body. The parasites measured an average of 40.7 μm in length (range , SD04.8) and 2.3 μm in width (range , SD00.3), based on measurements of 30 organisms. This is smaller than that reported by Roudabush and Coatney (1937), where the average length was 60.9 μm (range ) and average width was 3.5 μm (range ). Moreover, these authors reported a distinct broad undulating membrane and free flagellum (Fig. 1f), whereas we could not discern a flagellum on the parasites we observed, and a membrane was rarely seen (Fig. 1b). Also of note was the lack of variability in parasite appearance, either within individual blood films or across all infected salamanders. Roudabush and Coatney (1937) indicated that at least three forms were present in the infected hellbender they examined; there were thin as well as stout specimens (Fig. 1f), whereas all forms in the current study were long and slender (Fig. 1a e). Given the precarious conservation status of eastern and Ozark hellbenders (Wheeler et al. 2003; Foster et al. 2009; Burgmeier et al. 2011a), any pathogen that could negatively affect their populations should be closely monitored. While there is no information on the pathogenicity of T. cryptobranchi, in other vertebrates, some (but not all) trypanosome infections can negatively impact individual health (e.g., Muhammad et al. 2007). For example, an outbreak of Trypanosoma evansi killed 51 % of a population of horses in Brazil (Silva et al. 1995), Trypanosoma vivax is known to cause anemia and weight loss in dairy cattle (Silva et al. 1999), and in humans, Trypanosoma cruzi is a principal cause of morbidity and mortality on the South American continent (e.g., Kayama and Takeda 2010), while Trypanosoma brucei is a debilitating infection in Africa (e.g., Smith et al. 1998). Furthermore, even if outward health effects are not apparent, subclinical infections could predispose individuals to increased mortality (Smith et al. 2008), reduced fecundity (Goossens et al. 1997) or reductions in immunocompetence (Rurangirwa et al. 1983). We also note that this represents the first reported case of trypanosome infections that we are aware of from the eastern part of the range of C. alleganiensis alleganiensis. While Roudabush and Coatney (1937) did not report where their infected hellbender was captured, they indicated it was caught locally and these authors were located in Ames, IA, which historically contained small populations of eastern hellbenders (Bishop 1943) and is the apparent western limit of the range of this species. The discovery of this parasite in the eastern portion of the hellbender range emphasizes that further monitoring must be done to determine if this pathogen is spreading among hellbenders and whether it adversely influences their health and survival. Acknowledgments We thank Dr. Michael Yabsley for his help in identifying the hellbender parasite. We also thank Dr. Sarah DuRant, Mark Hepner, Brittney Hopkins, and Cathy Bodinof for assistance in capturing the hellbenders. Cathy Bodinof also provided helpful comments for improving the manuscript. 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